Single molecule fluorescence measurements are used to probe the effects of GM1 in DPPC monolayers. Langmuir-Blodgett films of GM1 and DPPC were doped with, similar to 10(-8) mol % of the fluorescent lipid probe, BODIPY-PC, and transferred onto glass substrates at 23 mN/m. As shown previously, the individual orientation of each BODIPY-PC probe in the membrane can be measured using defocused polarized total internal reflection fluorescence microscopy, revealing changes in film properties at the molecular level. Here, BODIPY-PC tilt angle histograms are used to characterize the effects of GM1 in DPPC films from 0.05 to 100 mol % GM1. At high GM1 levels (>5 mol % GM1), trends in the single molecule measurements agree with previous bulk measurements showing the turnover from condensing to expanding influence of GM1 at 15-20 mol %, thus validating the single molecule approach. At biologically relevant, low concentrations of GM1 (<5 mol % GM1), where bulk fluorescence measurements are less informative, the single Molecule measurements reveal a marked influence of GM1 on film properties. The addition of trace amounts of GM1 to DPPC films leads to an expansion of the film which continues to 0.10 mol % GM1, above which the trend reverses and the condensing effect previously noted is observed.